spinal cord repair strategies

Since ancient times it has been recognized that injury to the spinal cord can result in dramatic disability, including both negative symptoms (such as loss of voluntary move-ment and tactile sensibility) and positive symptoms (such as chronic pain and spas-ticity). Although many patients show some spontaneous recovery after injury, most patients with significant cord damage have permanent symptoms. Moreover, damage to the spinal cord, whether caused by injury or disease, cannot currently be repaired by any therapy. Yet there is now considerable optimism among neurobiologists that effective clinical therapies are within reach. This optimism is prompted on the one hand by a great increase in our understanding of the consequences of spinal cord injury (SCI) and the reasons for failure of spon-taneous repair, and on the other hand by an increasing number of treatments that have promoted functional improvements in experimental models of SCI (with func-tional outcome being assessed in several ways) (BOX 1). Yet, surprisingly perhaps, the mechanism(s) by which different experi-mental treatments promote functional improvement is in most cases undeter-mined. It is becoming clear that there are several distinct mechanisms that could underlie functional recovery, and that there is a considerable academic as well as practi-cal value in understanding their range and scope.

Traumatic SCI leads to a series of reactive changes. First, the injury could directly dam-age the cell bodies and/or processes of neu-rons. The cells that are damaged might die and, as far as is known, they are not replaced (although the adult spinal cord does contain stem cells). However, the functional conse-quences of this neuronal loss are typically modest. Often, the damage to spinal axons is much more important1. The spinal cord is segmentally arranged and the sensory, motor and autonomic functions of each segment depend crucially on connections with supraspinal sites for all conscious or voluntary actions (FIG. 1). Damage to these connections leaves spinal segments caudal to the lesion site partially or totally isolated from the brain, which has debilitating conse-quences. The distal axon segment (the part isolated from the neuronal cell body) retracts from postsynaptic neurons and undergoes Wallerian degeneration2, and although the proximal segment typically survives, it does not spontaneously regenerate. The injured axon also encounters a series of inhibitory cues within the injured CNS neuropil, which further prevent a successful regenerative response (BOX 2). An implicit assumption of much SCI research, at least until recently, has been that the major goal is to induce damaged axons to regrow, to reconnect to appropriate targets and thereby restore func-tion (as is indeed possible in the PNS). Here, we examine the extent to which this has

proved possible. In general, however, there is a lack of clear evidence that the regeneration of lesioned axons underlies significant func-tional improvements induced by experimen-tal treatments. This has led to an examination of alternative putative repair mechanisms, evidence for which is discussed below.

Regeneration of damaged axonsA major focus of SCI research has been to overcome the failure of axons to regenerate (FIG. 2). The three main aims of this research are: to initiate and maintain axonal growth and elongation; to direct regenerating axons to reconnect with their target neurons; and to reconstitute original circuitry (or the equivalent), which will ultimately lead to functional restoration. Although many inter-ventions have now been reported to promote functional recovery after experimental SCI, none has fulfilled all three of these aims. In fact, only the first aim has been extensively studied and convincingly demonstrated.

Axon regeneration after SCI. There is now a wealth of data demonstrating axon regenera-tion after experimental intervention. For example, strategies aimed at overcoming inhibitory environmental cues have resulted in significant regeneration of lesioned axons through and beyond sites of SCI. These strategies include the neutralization of myelin inhibitors3,4 and the degradation of inhibitory components of the glial scar 5,6. Axon regeneration after SCI has also been demonstrated following the administration of growth-promoting molecules, which make the lesion environment more permis-sive to growth. This includes the provision of exogenous neurotrophic factors7–9 or the manipulation of pro-regenerative neuronal signalling pathways10,11, as well as several cellular transplantation strategies such as genetically modified fibroblasts8,12, Schwann cell bridges13, olfactory ensheathing cells (OECs)14 and stem cells15. Furthermore, combination therapies aimed at targeting multiple factors have also been shown to act synergistically to enhance the extent of axon regeneration after SCI (BOX 3).

Although this is an impressive (and grow-ing) list, there are at least three important caveats to these studies. First, the contribution

O P I N I O N

Spinal cord repair strategies: why do they work?Elizabeth J. Bradbury and Stephen B. McMahon

Abstract | There are now numerous preclinical reports of various experimental treatments promoting some functional recovery after spinal cord injury. Surprisingly, perhaps, the mechanisms that underlie recovery have rarely been definitively established. Here, we critically evaluate the evidence that regeneration of damaged pathways or compensatory collateral sprouting can promote recovery. We also discuss several more speculative mechanisms that might putatively explain or confound some of the reported outcomes of experimental interventions.

644 | AUGUST 2006 | VOLUME 7 www.nature.com/reviews/neuro

PERSPECTIVES

© 2006 Nature Publishing Group



of regeneration to functional recovery is in most cases unknown. Second, the proportion of fibres that regenerate is always small. Third, the distance regrown is modest, certainly in the context of human spinal cord anatomy.

Regarding the first point, in most cases the best evidence is simply that the degree of regeneration observed is correlated with a concurrent recovery of behavioural func-tion. However, in individual experiments significant regenerative responses might be correlated with other adaptive mechanisms. In many cases it is also possible that what have been perceived as regenerating axons are, in fact, sprouting spared axons. Some studies have attempted to provide stronger evidence that regeneration is at least in part responsible for the observed func-tional recovery. For example, the functional improvements that were observed following treatment with IN-1 (an antibody that neutralizes the inhibitory myelin-associated protein Nogo-A) after SCI were abolished

following removal of the sensorimotor cor-tex, suggesting that the recovery depended on the regrowth of corticospinal tract (CST) axons3. Even here, the possibility remains that plasticity of brainstem and/or sensory systems contributed to the observed recovery (see below). Perhaps the best evidence comes from re-lesion experiments. Liu et al.12 transplanted genetically modi-fied fibroblasts to a lesion site to promote rubrospinal tract regeneration, and showed that a re-lesion rostral to the original lesion/transplantation site abolished the recovery of forelimb use in a vertical exploration task. However, although this indicates that recov-ery depended on the presence of the trans-planted tissue, it still does not determine the mechanism(s) by which the presence of a transplant can enable functional repair. Interestingly, re-lesioning can produce a more severe functional impairment than a single lesion alone16. This illustrates some of the potential problems of re-lesion experi-ments — that the re-lesion could be more

extensive than intended (‘to be safe’) and might thereby involve other pathways; or the abolition of function could be confused with oedema, spinal shock or inflammation-mediated conduction block that could occur as a result of the second lesion.

The number of regenerating axons fol-lowing experimental intervention is typically low, and at best amounts to only a small percentage of the original fibre tract. The majority of studies have focused on CST regeneration and have estimated success using anterograde or retrograde tracing techniques. Typically, 2–10% of CST axons are reported to regenerate. For example, as few as 5% of CST axons were induced to regenerate following Nogo-A neutraliza-tion, yet an almost complete recovery was observed in contact-placing responses and some aspects of locomotor function in treated animals3. A similar proportion has been observed in studies investigating rubrospinal tract regeneration, with typical estimates amounting to ~7% (REF. 12). It is worth comparing these results with the far superior regenerative capacity of the adult PNS (in which ≥90% of damaged axons regenerate under optimal conditions) or, of course, the targeted growth that occurs during development.

An important question, therefore, is whether these modest numbers of regen-erating axons can support the observed functional improvements. A priori, this might seem unlikely, but there is some evidence to the contrary. Raisman’s group have used electrolytic lesions to interrupt CST axons in adult rats14,17. They found that to achieve long-lasting deficits in a forepaw reaching task, ablation of the entire tract was required; when as little as 1–2% of the CST was spared, performance on this task recovered. Although in this case the lack of functional deficits

Box 1 | Measures of spinal cord injury outcome

The functional consequences of experimental spinal cord injury (SCI) have been assessed in many ways. One commonly used behavioural measure is the Basso, Beattie and Bresnahan (BBB) Locomotor Rating Scale89, which scores various aspects of hindlimb function during spontaneous locomotion and assigns a number from 0 to 21 to the experimental animal (with a score of 0 assigned for no observable hindlimb movement and 21 for normal locomotion). Many other forms of sensorimotor assessment have been used to measure functional outcomes following SCI and treatment interventions including error rates of beam or grid crossings, rope climbing, swimming tasks, adhesive tape removal, analysis of footprints and gait5,8,16,37,90. Measures of skilled forepaw use are often used as indices of corticospinal tract (CST) function14,37,91. There are batteries of tests used to assess sensory function, particularly sensitivity to painful stimuli88,92,93, but these are difficult to interpret as they virtually all depend on a motor end point (for example, paw flexion withdrawal) that could of course be compromised by the SCI. Autonomic functions have been less studied in experimental SCI models (with some exceptions; see REFS 94–96), although bladder and sexual dysfunction, and dysregulation of blood pressure and heart rate are considerable clinical problems in SCI58,97–99. The sensitivity and reproducibility of these tests differ. An improvement in performance on any of these tasks is usually taken as evidence for some repair after SCI but, as discussed here, the mechanisms underlying these improvements are often poorly understood.

Glossary

AllodyniaPain from stimuli that are not normally painful.

Chondroitinase ABC(ChABC). A bacterial enzyme that cleaves the sugar (glycosaminoglycan) chains from proteoglycan molecules, rendering them less inhibitory to growth.

Contact-placing responsesIn response to light contact of the foot an animal will lift its limb and place it on a surface for support.

Glial scarFollowing CNS injury, activated glial cells form a meshwork of interweaving processes that surround the lesion site. Scarring is important for sealing the wound but can also act as an impenetrable barrier to regeneration.

H-reflexA monosynaptic reflex elicited by electrically stimulating a nerve with an electric stimulus.

Immediate-early genesA family of genes that share the characteristic of having their expression rapidly and transiently induced on stimulation.

PreconditioningA protective effect from injury achieved by a previous insult (thought to be mediated by pro-regenerative changes in the cell body triggered by the insult).

PyramidotomyTransection of the corticospinal tract (CST) at the level of the medullary pyramids in the brainstem. A unilateral pyramidotomy lesions the CST on one side, leaving the

other side intact (thereby denervating one side of the spinal cord).

RhizotomyAn injury to the spinal dorsal roots that results in an interruption of sensory input from the PNS into the spinal cord.

Transcranial magnetic stimulationInvolves creating a strong localized transient magnetic field that induces current flow in underlying neural tissue, causing a temporary change in activity in small regions of the brain.

Wallerian degenerationDegeneration that occurs after axonal injury in the distal segment of a nerve fibre — the part no longer connected to the cell body.

P E R S P E C T I V E S

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was due to the sparing of a spinal projection (which makes appropriate connections) rather than the regeneration of lesioned axons (which might not), these observations nonetheless imply that regeneration of a small percentage of axons and restoration of their original circuitry could potentially lead to a significant recovery of function. Similarly, Wall et al.18 showed that more than 90% of dorsal column fibres needed to be lesioned for clear deficits in tactile sensory function to arise, and Windle et al.19 claimed that as little as 10% of spinal white matter tracts were sufficient to permit spontaneous walk-ing without external support in adult cats. Furthermore, this phenomenon was classi-cally demonstrated in human patients when large portions of spinal tracts were transected owing to intractable pain problems without causing disturbances in motility

20.The distance that central axons can

regenerate is also limited. Most studies have focused on rodents, and, even with the most successful interventions, regenerated axons are most numerous in the few millimetres caudal to the lesion, and longer regenerating axons become progressively rare. In the rat spinal cord, each spinal segment is typically 2–3 mm in length. The proportion of lesioned axons induced to regenerate at least two spinal segments (that is, enough to be of clear functional use) is typically less than 10% (REFS 3–5). Again, most of these data relate to the CST, although the more limited data available for other projections such as dorsal column sensory fibres7,9,10 or the rubrospinal tract12 are not dissimilar. Some studies have assessed regeneration in terms of the longest single regrown axon. In the rat, this is typically 10–15 mm. A crucial question — to which we do not have the answer — is whether these distances are absolute or rela-tive. That is, if these treatments were applied to human spinal cord, would they induce growth of up to approximately one or three spinal segments?

Evidence for functional connectivity. So far, few studies have shown that regenerating axons actually make functional postsynaptic connections. Perhaps the most robust way of demonstrating such connectivity is to perform an electrophysiological analysis. For example, Ramer et al.21 showed that regen-erating sensory axons made functional con-nections with dorsal horn neurons. Multiple rhizotomy of the cervical dorsal roots led to reduced sensory input to the spinal cord, and treatment with neurotrophic factors resulted in a re-emergence of postsynaptic activity in the dorsal horn in response to peripheral

nerve stimulation. Re-injury of the dorsal root abolished dorsal horn activity. A con-current recovery in sensitivity to mechanical and thermal noxious stimuli applied to the forepaw was observed, thereby providing convincing evidence that regenerated axons had made functional connections with target neurons, leading to a re-establishment of ‘normal’ circuitry and recovery of sensation

in the denervated forepaw. Similarly, Steinmetz et al.22 demonstrated a recovery of the H-reflex response after rhizotomy and treatment with chondroitinase ABC (ChABC) combined with a preconditioning nerve injury. H-reflex recordings disappeared following acute resection of the dorsal root, indicating that functional connectivity of regenerated axons had occurred.

Figure 1 | Structure of the spinal cord. The spinal cord is responsible for transmitting signals between the brain and body. It is encased in vertebral bone and is segmentally arranged. The seg-ments are grouped into 4 major divisions: cervical (C; 8 segments), thoracic (T; 12 segments), lumbar (L; 5 segments) and sacral (S; 5 segments). Each spinal segment makes connections with discrete body regions through projections running in the sensory and motor roots. The spinal segments are con-nected to the brain through a number of long axonal pathways that mostly run around the periphery of the cord. The main ascending pathways (which transmit sensory information to the brain) are indicated in red and the main descending pathways (which transmit motor information to the body) are indicated in blue. An injury to the spinal cord has devastating consequences owing to the disrup-tion of these signals passing between brain and body, resulting in essentially no feeling or control of function below the level of the injury. Therefore, the higher the injury the more severe the debilita-tion. For example, injuries occurring at the lumbar level can result in paraplegia, as well as sexual and bladder dysfunction; cervical injuries are more debilitating and can result in quadriplegia; and high cervical injuries (such as the injury suffered by Christopher Reeve, level C2) can impair breathing function and lead to dependence on a ventilator. Anatomical image adapted, with permission, from REF. 132 © (1996) Appleton and Lange.







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This type of analysis (electrophysiologi-cal assessment of functional connectivity then re-lesion) is, for practical reasons, more difficult to apply after the lesion-ing of CNS tracts, but there are already several examples of what might be done. For instance, Bradbury et al.5 electrically stimulated corticospinal neurons below the SCI level in lesioned animals treated with ChABC, and found a partial restoration of postsynaptic activity. These responses had longer latencies than in unlesioned animals, consistent with poorly myelinated regen-erating axons. Furthermore, when the CST was acutely re-lesioned, this activity was abolished, providing evidence that func-tional reconnections had been formed by regenerating CST axons. Other approaches include the use of transcranial magnetic stimulation to activate corticospinal neurons, coupled with electromyographic recordings of, for example, hindlimb musculature23. Immunohistochemical techniques — such as assessing for the presence of the synaptic marker synaptophysin, or activity-depend-ent expression of the immediate-early gene c-fos in postsynaptic cells — might also provide strong supporting evidence of func-tional connectivity

24,25. With an increasing number of experimental treatments report-ing pro-regenerative effects, it is vital that this question of functional connectivity is more comprehensively addressed.

Anatomical plasticityThe term ‘neuronal plasticity’ is used to describe a wide range of phenomena. One example relates to brain development, where multiple synaptic contacts are formed and experience determines which connections

will be strengthened and which will be pruned. Another form of plasticity, which we consider here, is an adaptive reorganization of the neural pathways occurring after injury that acts to restore some of the lost function. We examine the hypothesis that functional recovery after SCI arises from the collateral sprouting of either damaged or spared path-ways, which leads to the creation of novel neuronal circuits (FIG. 3). We first discuss evidence for spontaneous neuronal plasticity after SCI. We then consider the growing evidence that several treatments for experi-mental SCI act to enhance this process.

Spontaneous neuronal plasticity after SCI. Although many SCIs lead to permanent dis-abilities, when the SCI is incomplete there is frequently a period of significant functional improvement. This is seen in many spe-cies, albeit with different time courses26–28. Although this recovery was originally described as recovery from ‘spinal shock’, it is perhaps more useful to describe it in terms that have mechanistic meaning. One contributing mechanism appears to be ana-tomical plasticity

29. The spinal shock phase in humans is well defined, and lasts for sev-eral weeks or more. In animals, it is typically shorter, lasting for hours or days depending on the species30. It might be due in part to the loss of tonic descending excitation (for example, by the serotonin-mediated system), as shown by the fact that even spinal

Box 2 | Inhibitory cues present after spinal cord injury

Axonal regeneration following spinal cord injury (SCI) is limited partly because CNS neurons have a poor intrinsic capacity for growth, but also because injured axons encounter a series of inhibitory factors that are non-permissive for growth.

Myelin inhibitors. The first CNS neurite growth inhibitor to be discovered was an inhibitory protein fraction of CNS myelin100, now known as Nogo-A101–103. Nogo-A is inhibitory to neurite outgrowth in vitro101–103, and several studies in vivo have observed regeneration, plasticity and functional recovery following treatments that neutralize Nogo-A3,37,104 or block Nogo receptor function4,23,41. Although evidence from Nogo-A knockout mouse studies remains controversial105, there is nonetheless a wealth of evidence that this molecule is a potent neurite growth inhibitor106,107. Other identified CNS myelin-associated inhibitors include MAG108 (myelin-associated glycoprotein) and OMgp109 (oligodendrocyte myelin glycoprotein). The effects of neutralizing these inhibitors in vivo have not been extensively explored.

Chondroitin sulphate proteoglycans. Proteoglycans are extracellular matrix molecules that consist of a protein core with attached sugar moieties called glycosaminoglycans (GAGs). They are characterized by their GAG composition as chondroitin-, heparin-, keratin- and dermatin-sulphate proteoglycans. Chondroitin sulphate proteoglycans (CSPGs) form a large family including neurocan, versican, aggrecan, brevican, phosphacan and NG2. CSPGs are found throughout the adult CNS and are strongly upregulated following SCI, where they contribute to the glial scar44,110–112. They are inhibitory to neurite outgrowth in vitro113–115 and in vivo116. Disruption of the GAG portion of CSPG molecules makes inhibitory substrates more permissive to growth117 and can promote regeneration and functional recovery after CNS injury5,118,119.

Although these two classes of inhibitors have been the most widely studied, several others, including the semaphorins120 and ephrins121, are found in the CNS and might have a role in limiting growth after SCI.

Figure 2 | The major functional deficits associated with spinal cord injury arise from the interrup-tion of long ascending and descending spinal tracts. When the normal axons of long ascending and descending spinal tracts (a) are damaged (b), the distal segment undergoes Wallerian degeneration and the proximal segment is unable to successfully regenerate. Therefore, ascending (sensory) informa-tion no longer reaches supraspinal sites, and descending motor and autonomic systems are discon-nected from caudal spinal circuitry, including central pattern generators (CPGs), which are responsible for coordinated locomotor function. Much spinal cord injury research has focused on the possibility of inducing these damaged projection systems to regenerate and recapitulate original circuitry (that is, to restore the normal arrangement, as illustrated in c; regenerating axons are depicted in red).







monosynaptic reflexes are affected. A signifi-cant part of spontaneous functional recovery in SCI patients occurs 2–6 months after injury, and is closely linked to intensive reha-bilitative treatment. Here, the underlying mechanisms are likely to include neuronal plasticity and rearrangement.

Early studies demonstrated the capacity for functional compensation of descending pathways following injury. For example, compensatory plasticity of rubrospinal and bulbospinal tracts was found to mediate the recovery of forepaw function following CST lesion in cats31. Similarly, following a dorsal column lesion in rats, the spontaneous recovery of skilled reaching ability was found to be due to compensation by other sensori-motor pathways32. The importance of CST plasticity as a mechanism underlying such recovery has also been demonstrated. For example, Weidner et al.33 demonstrated that transection of the dorsally projecting CST led to spontaneous sprouting of the ventral CST projection, which was paralleled by a recovery of skilled forelimb use. This recov-ery was abolished by subsequent lesioning of the ventral CST. Remodelling of minor CST components (dorsolateral and ventral CST) after dorsal CST lesioning has also been recently demonstrated in a transgenic mouse in which the CST is exclusively labelled34.

Furthermore, Bareyre et al.35 demonstrated that spontaneous recovery of function after a dorsal hemisection injury involves extensive reorganization of spinal circuits. They found that some of the transected CST axons (that would normally innervate lumbar segments) sprouted into the cervical cord to innervate propriospinal neurons. Some of these con-nections were transient (those with short propriospinal neurons that did not bridge the lesion), whereas others were maintained in the long term (those with long propriospinal neurons that circumvented the injury). This led to a novel, indirect motor pathway to lumbar motor circuits. Importantly, the functionality of these connections was confirmed electrophysiologically and corres-ponded with anatomical evidence of transy-naptic connectivity as well as improvements in hindlimb function. Unilateral transection of the CST in the brainstem significantly reduced the improvements in hindlimb function, indicating that recovery depended on the new CST projections. The molecular events that direct this remarkably specific and novel change are not clear. Therefore, in several cases of experimental SCI, functional improvements occur over time in lesioned animals without any treatment intervention. Is it possible that some treatment interven-tions enhance this type of spontaneous

recovery by increasing the plasticity of intact (and injured) systems, rather than by frank regeneration? Evidence that this is indeed the case is discussed below.

Plasticity induced by experimental treatments for SCI. There is now strong evidence that two types of treatment that lead to functional improvements in experimental SCI can also induce significant anatomical plasticity. The first treatment type targets the inhibitory components of CNS myelin. For example, collateral sprouting of the unlesioned CST into the denervated spinal cord has been demonstrated following unilateral pyramidotomy and IN-1 treatment. This compensatory sprouting was accom-panied by the recovery of forelimb motor behaviours on the denervated side36,37. Furthermore, after bilateral pyramidotomy, reorganization of the rubrospinal tract has been observed following IN-1 treatment, with increased innervation of ventral horn motor neurons that normally receive CST input; this too was associated with recovery of precision movements of the forepaw38. Functional connectivity of these novel rubrospinal projections was confirmed electrophysiologically following red nucleus microstimulation39.

Further evidence comes from observa-tions of corticospinal and raphespinal fibre sprouting after dorsal over-hemisection injury and blockade of Nogo receptor (NgR) function. This sprouting was associated with a recovery in locomotor perform-ance23,40,41. In an earlier study, delivery of the peptide fragment NEP1–40 (to block NgR signalling) led to functional recovery in hemisected rats and some regeneration of CST axons4. However, the functional recovery was observed as early as 2 days post-lesion, and it is unlikely that regenerat-ing axons could have reformed functional postsynaptic connections in that time. It is therefore probable that the early recovery was mediated by plasticity mechanisms.

Corollary evidence that inhibitory com-ponents of CNS myelin restrict plasticity has come from stroke models. Partial recovery of function frequently occurs after cortical ischaemic injury, and this is largely attributed to the plasticity of axonal connections. Anti-Nogo therapy has been shown to improve functional recovery in experimental stroke models, and this too has been attributed to the reorganization and plasticity of unlesioned cortical areas42,43. Therefore, manipulation of myelin inhibitors after injury can unmask the capacity of the adult CNS for reorganization. However, the

Box 3 | Combination therapies act synergistically to enhance regeneration

As spinal cord injury (SCI) is a multifaceted problem it is widely believed that combinatorial treatments will be required when translating experimental research findings to clinical therapies. Several studies have now attempted to address this issue by targeting multiple factors. For example, treatment with the enzyme chondroitinase ABC (ChABC), which breaks down inhibitory sugar chain components of CSPG molecules, promotes regeneration of lesioned axons in the brain118 and spinal cord5,6. This treatment has now been used in combination with other interventions and has been shown to enhance growth across the dorsal root entry zone in combination with an inflammation-induced preconditioning treatment22; to enhance axonal regrowth beyond the graft-host interface of Schwann cell-seeded guidance channels in the injured spinal cord122; to promote migration of transplanted neural stem/progenitor cells and fibre outgrowth following spinal contusion injury123; to improve regeneration and locomotor recovery when combined with transplanted Schwann cells and olfactory ensheathing glia following cord transection124; and, when combined with lithium chloride, to improve rubrospinal tract regeneration and forelimb use after spinal hemisection125.

There is also a history of studies that have combined growth factor treatment with cellular grafting techniques to maximize the growth potential of injured spinal axons. For example, infusion of neurotrophins has been shown to enhance regeneration into Schwann cell grafts126. A more sophisticated approach has been to genetically modify cellular graft tissue (such as fibroblasts and Schwann cells) prior to transplantation to secrete neurotrophic factors, thereby providing injured axons with a cellular bridge and a source of trophic support8,12,127 (for reviews, see REFS 128,129). Similarly, other studies have combined treatments that induce a regenerative state in the cell body (by elevating cyclic AMP levels) with treatments that promote regeneration of the lesioned axons, such as neurotrophic factors130 and Schwann cell transplants131.

The above studies all show enhanced axonal regeneration when two or more treatments are combined. Many of these studies also provide a convincing demonstration of functional recovery. Therefore, as with individual therapeutic treatments (discussed in the main text), the principal mechanism proposed for the synergistic effects of combination treatments has been that they increase the extent of axon regeneration. However, evidence that regeneration is indeed the cause of functional recovery has not been conclusively demonstrated.







a b

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extent to which this plasticity is responsible for functional improvement has not been established in most studies.

Chondroitin sulphate proteoglycans (CSPGs) also contribute to the inhibitory environment of the CNS, particularly after injury

44. CSPGs are increasingly being seen as molecules that regulate plasticity in the adult CNS (for a review, see REF. 45). Key evidence first came from studies in the brain; for example, high levels of CSPGs are present in perineuronal nets46, and disruption of these using ChABC can restore synaptic plasticity in the visual cortex of adult rats to a level normally seen only during develop-ment

47. Degradation of CSPGs by ChABC treatment also leads to sprouting of intact Purkinje axons in the cerebellum48 and pro-motes sprouting of undamaged retinal affer-ents into the denervated superior colliculus following a partial retinal lesion injury

49.Recent evidence shows that CSPGs can

also regulate plasticity in the spinal cord. In recent work from our laboratories, we have observed sprouting of both intact and injured descending pathways as well as intact sensory afferents following SCI and treatment with ChABC (A. W. Barritt et al., unpublished observations). In all cases, sprouting fibres were observed in aberrant locations close to the SCI (within one or two segments) in

areas of degeneration and CSPG degradation. Although these data provide evidence that CSPGs are important for restricting the plas-ticity of adult spinal systems, the functional role of these sprouting fibres is less clear. However, a recent study has demonstrated functional collateral sprouting of forelimb sensory afferents into partially denervated brainstem nuclei following ChABC injec-tions50. This study combined anatomical tracing techniques with receptive field mapping to demonstrate sprouting of intact afferents and an expansion of receptive fields after CSPG degradation. However, it did not determine whether such sprouting could affect forelimb function. By contrast, we have found that terminal field expansion within the spinal cord following ChABC treatment can result in a remarkable recovery of fore-limb function following denervation51. In a spared-root injury model (in which all the dorsal roots from C5–T1 are rhizotomized, with the exception of C7), little activity was recorded in the C7 spinal cord following stimulation of the C7 dorsal root (deter-mined by recording cord dorsum potentials, focal field potentials and single units in the dorsal horn). A single intraspinal injection of ChABC restored postsynaptic responses to levels that were indistinguishable from those observed in uninjured control animals and

led to a complete recovery on several tests of sensory function of the denervated forelimb. These data provide robust evidence that degrading CSPGs within the spinal cord can lead to a functional reorganization of dorsal horn input, and that novel connections are organized in a functionally meaningful way.

The results of these studies suggest that CSPGs have a similar role to myelin inhibi-tors in suppressing sprouting responses of injured and intact systems after injury. Targeting both myelin and CSPG inhibitors might be a future strategy to consider for maximizing the potential for sprouting or plasticity after SCI.

It should be noted that in addition to having beneficial effects, increased sprouting can also lead to abnormal connections being made, with potentially detrimental conse-quences. For example, increased primary afferent sprouting in the spinal cord of experi-mental animals has been associated with pain and autonomic dysreflexia52,53. In one study, the beneficial effects on motor function observed following neural stem cell trans-plants were limited by the development of allodynia, which was associated with aberrant sprouting54. The outcome was improved by differentiating the cells prior to transplanta-tion, but this study highlights the importance of balancing potentially opposing influences. Pain, autonomic dysreflexia and spasticity are common symptoms in patients with SCI

55–59 that could potentially be increased if abnor-mal connections were formed. Therefore, targeted treatments aimed at minimizing detrimental sprouting and optimizing ben-eficial sprouting will be a future goal for the treatment of patients with SCI.

Other mechanismsAlthough there is some (albeit limited) evi-dence that regeneration and anatomical plasti-city of connections contribute to functional recovery after SCI, there are several less well-explored, but entirely plausible, putative processes that might also contribute to repair. If these, or indeed any other mechanisms, can contribute to functional recovery from SCI, there is a self-evident value in optimiz-ing their effects. Establishing that some or all of them do not contribute to functional improvement might also be important in removing experimental confounds. Some of these alternative mechanisms are discussed below and illustrated in FIG. 4.

Functional reorganization of spinal circuits. After complete spinal cord transection at thoracic levels, all voluntary control of hindlimb locomotion is lost. However,

Figure 3 | Restoration of function after spinal cord injury might arise from anatomical plasti-city of damaged or spared connections. a | Collateral sprouts (illustrated in red) might form from long descending pathways that bypass the lesion site and activate spinal circuits more effectively or activate novel circuits, or from damaged or intact descending projections that activate local proprio-spinal neurons that bypass the injury site and form a novel descending system. b | Ascending sensory projections might also form collateral sprouts, which activate spinal circuits more effectively. CPG, central pattern generator.







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Treatment Treatment No treatment

Effects of treatment owing to changes in motivational state

there is a considerable body of work that shows that pharmacological manipula-tions of such isolated spinal segments can enhance locomotor activity in various ways and species. For example, NMDA (N-methyl-d-aspartate) receptor agonists and manipulations that enhance serotonin or noradrenaline levels can act synergisti-cally to induce treadmill walking (in which body weight is supported) in spinalized cats (for a review, see REF. 60). These effects are present almost immediately after treatment and so cannot represent anatomical regen-eration or sprouting. Rather, they unmask or enhance existing neuronal mechanisms. But in so doing they reveal a major shift in the functional capabilities of the spinal seg-ments caudal to injury. Following partial, rather than complete, SCIs this enhance-ment of locomotor function might greatly increase voluntary control of locomotion, but the mechanism would have nothing to do with repairing the original damage. Treadmill training, which is used in many SCI clinics with great success61,62, might promote this form of plasticity.

Furthermore, many of the experimental treatments that are reported to improve

function after SCI (such as neurotrophic factor treatment) are likely to alter neuro-transmitter levels in the spinal cord63, or affect synaptic strengths in other ways64. Another example is the powerful hetero-synaptic facilitation of dorsal horn neuron responsiveness that is induced in seconds by simple repetitive activity in small diam-eter sensory neurons (for a review, see REF. 65). This phenomenon potentiates spinal reflex excitability, thereby pot-en tially confounding the analysis of sensorimotor or sensorivisceral function. The spinal facilitation depends in part on glutamate acting at NMDA receptors and brain-derived neurotrophic factor acting at TrkB receptors, and therefore could plausi-bly be modulated by several experimental ‘treatments’ for SCI.

Remyelination of fibre tracts. SCI results not just in Wallerian degeneration of damaged axons, but also in demyelination of some intact axons66,67, and the functional impair-ments following experimental SCI have been correlated with the degree of demyelin-ation68. These demyelinated axons have less secure transmission of action potentials

and complete failure when even moderate lengths of axon are demyelinated. Caudal spinal segments are therefore functionally disconnected from supraspinal centres. The loss of oligodendrocytes that leads to demyelination might itself be modified by some treatments for SCI, such as neurotrophic factor delivery; moreover, established demyelination might be reversed by these (or other) treatments69. There is also evidence that enhancing nerve conduction through areas of demyelination by increasing the duration and safety of action potential transmission could improve function after SCI70,71. Therefore, some treatments might produce a marked improvement in function without actually promoting any regenera-tion. Again, several of the treatments for SCI (most obviously the transplantation of stem cells, OECs or other glia17,72,73, but conceiv-ably also some treatments targeting puta-tive myelin inhibitors) could affect either de myelination or remyelination.

Amelioration of secondary damage. In humans and other species, many SCIs are compounded by secondary damage in the form of cavity formation or extension74–76 and

Figure 4 | Various well-established phenomena might contribute to functional recovery from spinal cord injury after experimental inter-ventions. a | Rapid changes in synaptic efficacy can be triggered in the spinal cord by activity, neurotrophic factor treatment and manipulation of neurotransmitter systems, among others. Some of these changes are known to be associated with altered functional responses such as locomotion. b | Spinal cord injuries (SCIs) are known to be associated with demyelination of some intact axons. Several treatments aimed at promot-ing regeneration might in fact reduce the degree of demyelination or promote recovery from it, thereby accounting for some of the improved function. c | SCI is frequently associated with a slowly increasing area of

secondary damage. Several experimental interventions could interfere with this secondary damage and so promote an apparent recovery. d | Another speculative mechanism of functional recovery relates to an altered motivational state. One example is putative analgesic actions. Many SCIs lead to chronic pain, triggered at least in part by local inflam-matory processes. Many experimental treatments for SCI could affect these inflammatory or other pain-producing processes and in so doing increase functional capabilities, illustrated here as improvements on the Basso, Beattie and Bresnahan (BBB) Locomotor Rating Scale89. The mechanisms illustrated are not comprehensive, but they represent plausible options that have rarely been considered. CPG, central pattern generator.







a relatively slow-developing penumbra of cell death spreading from the injury site77,78. A pronounced inflammatory reaction also contributes to secondary pathology after SCI77,79. The causes of this secondary damage are not well understood, and it is therefore easy to speculate that multiple treatments for SCI might interfere with this phenomenon. Although several experi-mental treatments have been specifically targeted to minimize secondary damage, in particular by modifying inflammatory responses76,80–82, it is also possible that other experimental treatments indirectly affect secondary pathology. Clearly, several factors (including neuroprotective factors, cellular transplantation and indirect effects on vascularization) are all potential modi-fiers of this process. It would be useful to develop well-validated methods to assess such secondary processes.

Altered motivational state. A more specula-tive explanation for functional recovery is that some treatments affect motivational or mood states. Such indirect actions on brain function are always possible if the treatment has systemic effects. However, one example of a local effect at or close to the lesion site that might affect motivation is a potential analgesic action. SCI leads to chronic pain problems in a large proportion of patients with SCI55. Experimental SCI in animals also produces altered pain sensitivity at and below the lesion site83. The molecular mechanisms underlying SCI-associated pain are poorly understood, but there is increasing interest in understanding them84–86. For example, pro-inflammatory cytokines released in the injured spinal cord from various immune and glial cell types are likely to be contributory factors87. Therefore, treatments that affect these processes might plausibly affect pain secondary to SCI. Indeed, treatment with the microglial inhibitor minocycline has recently been shown to attenuate the hyper-respon-siveness of lumbar dorsal horn neurons and hypersensitivity to painful stimuli that occurs following SCI88. Interestingly, the effects on locomotor performance were less clear, with a partial recovery reported (using Basso, Beattie and Bresnahan (BBB) Locomotor Rating Scale scores). Further exploration is needed to determine whether analgesic treatment (putatively a side effect of some SCI treatment interventions) affects the locomotor or other functional outcomes of animals with SCI. We speculate that many putative treatment interventions might have unexpected non-specific effects that could be responsible for the observed functional recovery.

ConclusionsThe literature on SCI suggests that several treatment interventions can promote regeneration of damaged axons. The degree of such regeneration remains modest, but might be sufficient to account for func-tional recovery. Compensatory collateral sprouting has also been observed to occur spontaneously after SCI, and this process can be enhanced considerably by several interventions. Unfortunately, the extent to which these two mechanisms account for the functional effects of a known treatment is still not fully established. There are other eminently plausible mechanisms that could contribute to functional recovery. These remain mostly untested at present, and those discussed here do not form an exhaustive list. The diversity of mechanisms that might promote recovery from SCI could increase the options for developing novel therapies, but it also makes it more important to iden-tify the mechanisms that are activated by any one treatment.

Elizabeth J. Bradbury is at the Neurorestoration Group, Wolfson Wing, Hodgkin Building, Guy’s Campus, King’s College London, London Bridge, London SE1 1UL, UK.

Stephen B. McMahon is at the Neurorestoration Group and the London Pain Consortium, Wolfson Wing, Hodgkin Building, Guy’s Campus, King’s College

London, London Bridge, London SE1 1UL, UK.

Correspondence to S.B.M. e-mail: [email protected]

doi:10.1038/nrn1964

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AcknowledgementsThe work of the authors is supported by grants from the Medical Research Council and the Wellcome Trust, whom we would like to thank. We would also like to thank T. Boucher and D. Bennett for advice on the manuscript.

Competing interests statementThe authors declare no competing financial interests.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneNgR | Nogo-A

FURTHER INFORMATIONBradbury’s homepage: http://www.kcl.ac.uk/depsta/biomedical/CARD/bradbury_profile.htmMcMahon’s homepage: http://www.kcl.ac.uk/biomedical/mcmahon/index.htmlAccess to this links box is available online.







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